Sydney O. Ugwu

Preparation, Characterization, and Stability of Liposome-Based Formulations of Mitoxantrone

The preparation, characterization, and stability of lyophilized liposome-based formulation of mitoxantrone was investigated. Mitoxantrone was entrapped inside small, unilamellar liposomes composed of dioleoylphosphocholine (DOPC), cholesterol, and cardiolipin. The mean vesicle size and drug entrapment efficiency of the liposomes were ~ 150 nm and ~ 99%, respectively. Less than 1% of drug was lost and mean vesicle size remained unchanged after sterile filtration. The pre-lyophilized (pre-lyo) formulations were characterized by a differential scanning calorimetric (DSC) method. Results showed that the glass transition temperatures (Tg′) increased as the molar ratios of sucrose:lipid and trehalose:lipid in the formulations were increased. The maximum Tg′ of the pre-lyo formulations containing 10:1 sucrose:lipid and trehalose:lipid molar ratios were − 37°C and − 41°C, respectively. After reconstitution of the lyophilized cake of the sucrose-containing formulation, the mean vesicle size was comparable to pre-lyo liposome size. In vitro release studies showed that less than 2% of mitoxantrone was released after an extensive dialysis against phosphate buffered saline (PBS) at 37°C, indicating that the mitoxantrone was highly associated and retained inside the liposomes. Short-term stability studies of the sucrose-containing formulations revealed that the reconstituted and eight-fold diluted formulations were stable for up to 8 hours at room temperature. Long-term stability studies of lyophilized liposomal mitoxantrone showed that the lyophilized formulation was stable for up to 13 months after storage at refrigerated condition.

Skin pigmentation and pharmacokinetics of melanotan-I in humans

A comparative pharmacokinetic trial was performed with a superpotent synthetic melanotropic peptide, [Nle4‐D‐Phe7]‐α‐MSH1–13 (melanotan‐I or MT‐I) given by three routes of administration. Plasma levels were measured by RIA and tanning was quantitated using serial reflectometry. Doses of 0·16 mg kg−1 were administered intravenously (IV) and orally (PO), and doses from 0·08 to 0·21 mg kg−1 subcutaneously (SC), in a randomized crossover fashion to three male volunteers over five consecutive days for 2 weeks (ten doses). The results indicate that the SC dose is completely bioavailable compared to the IV dose. No detectable drug levels were observed following PO dosing. The plasma half‐lives following SC dosing ranged from 0·07 to 0·79 h for the absorption phase and from 0·8 to 1·7 h for the β‐phase. Clearance ranged from 0·12 to 0·19 L kg−1 h−1 and 3·9% or less of the dose was recovered in the urine. Side‐effects were minimal, consisting of occasional gastrointestinal upset and facial flushing. Significant tanning of the forehead, arms, and neck was noted following IV or SC dosing. This effect peaked at 1 week following drug administration but was still present 3 weeks after completing the ten‐dose regimen. It is concluded that SC administration is an efficacious method of delivering melanotan‐I.

High-performance liquid chromatographic assay for melanotan-1 ([Nle4-DPhe7]α-melanocyte-stimulating hormone) in biological matrices

The overall objective of this research was to develop a sensitive, specific, and stability-indicating HPLC assay for the determination of the [Nle4-DPhe7]alpha-melanocyte-stimulating hormone analog known as Melanotan-1 (MT-1) in biological matrices, i.e., cell culture transport media and human plasma. Separation was accomplished isocratically within 8.0 min using a C8 reversed-phase column. The mobile phase consisted of 0.1 M phosphate buffer-acetonitrile (80:20, v/v) with 18 microliters/l triethylamine at pH 2.50. The flow-rate was 1 ml/min with detection at 214 nm. Standard curves (n = 5) were linear over the concentration range 100-1000 ng/ml. The precision, accuracy, intra- and inter-day variations were good with C.V.s typically within 8.7% for concentrations greater than 100 ng/ml. This method was applied to a study of the transport of MT-1 in the Caco-2 cell monolayer model.

Characterization of the complexation of diflunisal with hydroxypropyl-β-cyclodextrin

The equilibrium dialysis method was applied to the determination of drug cyclodextrin stability constants using diflunisal and 2-hydroxypropyl-beta-cyclodextrin (HPBCD) as a model system. Analysis of the data showed the existence of a linear Scatchard plot, indicative of the formation of a 1:1 diflunisal:HPBCD complex. The mean complexation constant (Kc) +/- S.D. was 3,892 +/- 360 M(-1). The stoichiometry of the complex was verified using the appropriate mass action law equation. The diflunisal:HPBCD complex was also investigated using titration microcalorimetry. A Kc of 3,394 M(-1) was obtained together with an enthalpy change (deltaH) of -20.76 kJ/mol(-1). The Kc values obtained here using the equilibrium dialysis and microcalorimetric methods were comparable to one reported previously using a potentiometric method (5,564 +/- 56 M(-1)).

An improved method for determination of acid dissociation constants of peptides

AbstractPurpose. The method described here enables the proton dissociation constants of several amino acid residues of a peptide to be determined simultaneously in aqueous solution without prior knowledge of the exact concentration of the peptide. Methods. The method used here employs a non-linear fitting program, the BEST program, or a linear least-squares method in combination with the BEST program. These methods are discussed in detail with an emphasis on the quality of the potentiometric titration data that are obtained. Two representative peptides, one with two proton dissociation constants (Kal, Ka2) and the other with four proton dissociation constants (Kal–Ka4) were used to illustrate the advantages and the limitations of these two complementary methods. Results. The pKa values of TVL, a schizophrenia-related tripeptide, were found to be 3.62 (±0.02) and 7.17 (±0.05); the pKa values of ELTLQE, a hexapeptide, were found to be 2.32, 3.77,4.58 and 7.74. Conclusions. The methods reported here are generally applicable to a variety of peptides. The possibility of integrating these procedures into a preparative chromatographic system for the “on-line” assessment of the pKa values of peptides during the purification stage is an attractive and novel feature of this method.

Absorption enhancement of melanotan-l: Comparison of the caco-2 and rat in situ models

AbstractThe overall objective of this research was to evaluate the potential of Melanotan-I (MT-I) for oral delivery in humans. An in vitro cell monolayer model (Caco-2) was used to screen the effect of certain absorption enhancers on the transport of MT-I. The enhancers used were dimethyl-β-cyclodextrin (DMβCD), sodium tauro-dihydrofusidate (STDHF), and aprotinin, a protease inhibitor. In addition, the effect of the most promising enhancers on the bioavailability of MT-I was deter-mined using an in situ closed loop rat model. In the Caco-2 monolayer model, coadministration of MT-I with aprotinin produced a 2.4-fold increase in the trans-port of MT-I. No significant enhancement was seen with either DMβCD or STDHF. Thus, inhibition of degradation of MT-I by proteases appears to be the most promising approach for delivering MT-I via the oral route. Finally, an attempt was made to compare the transport data from the Caco-2 model to bioavailability data in the rat model. The trends observed for both models we...

The Application of Equilibrium Dialysis to the Determination of Drug-Cyclodextrin Stability Constants

The equilibrium dialysis method was applied to the determination of drug-cyclodextrin stability constants using diflunisal and 2-hydroxypropyl-β-cyclodextrin (HPBCD) as a model system. Analysis of data showed the existence of a linear Scatchard plot, which is indicative of the formation of a 1:1 diflunisal:HPBCD complex. The stoichiometry of the complex was verified using the appropriate mass action law equation. The complexation constant (Kc was 3801 ± 541 M−1. The Kc obtained using the equilibrium dialysis method was comparable to that obtained using a potentiometric method (5564 ± 56 M−1)

Kinetics of degradation of a cyclic lactam analog of α-melanotropin (MT-II) in aqueous solution

Abstract The kinetics of degradation of MT-II in aqueous buffered solution was studied in order to facilitate the formulation of a stable oral dosage form. A stability-indicating high-performance liquid Chromatographic (HPLC) assay was used to measure the concentrations of MT-II remaining at various time periods. The rate of degradation of MT-II was studied as a function of pH, phosphate buffer concentration, temperature and ionic strength. Results indicated that the degradation of MT-II followed apparent first-order kinetics. The pH-rate profile showed that MT-II was most stable at approximately pH 5.0. Data obtained from this study also indicated that the degradation rate of this peptide was directly proportional to phosphate buffer concentration and temperature. The shelf-life of MT-II in aqueous buffer solutions at 25°C was 27 h. The activation energy was 7.5 kcal/mol. The degradation rate of MT-II appeared to be independent of the ionic strength of the aqueous buffered solution.

High-performance liquid chromatographic assay for the α-melanotropin[4,10] fragment analogue (Melanotan-II) in rat plasma

A high-performance liquid chromatographic (HPLC) procedure has been developed for the quantification of Melanotan-II (MT-II), a cyclic heptapeptide which promotes rapid tanning of the skin, in rat plasma. The method involves precipitation of plasma proteins followed by direct-injection HPLC with ultraviolet detection. Calibration curves were linear over the range 100-1000 ng/ml for rat plasma. The method is reproducible and reliable with a detection limit of 50 ng/ml in plasma. Within- and between-day precision and accuracy reported as coefficient of variation and relative error, respectively, were < 7%. The application of the assay was successfully demonstrated by quantifying the concentration of MT-II in rat plasma samples following an intravenous dose of 0.3 mg/kg.

Partitioning properties and degradation kinetics of the [Nle4-DPhe7]α-MSH analog Melanotan-I (MT-I)

Abstract The objectives of this research were to determine the partitioning properties and the solution stability of the [Nle 4 -DPhe 7 ]α-MSH analog called Melanotan-I (MT-I). The apparent partition coefficients (APC) were determined in two solvent systems, i.e. octanol-buffer and isooctane-buffer in order to calculate Δ Log P.C. (partition coefficient) values. The Δ Log P.C. values at pH 2.5 and 7.4 were −0.307 and −0.409, respectively. The solution stability of MT-I was determined by accelerated stability studies at 70°, 80° and 87°C at pH 7.4 and ionic strength (μ) of 0.17. The energy of activation ( E a ) was calculated to be 15.83 kcal/mol, resulting in an estimated shelf life of 40 days at room temperature. From the pH-rate profile, MT-I was observed to be relatively stable under acidic conditions and exhibited greater degradation under basic conditions. Both ionic strength (μ = 0.15 to 1.5, at 0.05 M buffer concentration) and phosphate buffer concentration (0.1–0.5 M, μ = 1.5) at pH 7.3 and 80°C had no effect on the degradation kinetics of MT-I.